The technical field of this invention is reconstruction of artificial organs by perfusing cultured cell populations into decellularized scaffolds formed from harvested animal or cadaver organs. The invention is particularly useful in constructing artificial kidneys for implantation.
Acute renal failure refers to the disruption of normal kidney function. This clinical condition arises due to a variety of mechanisms including infections, circulatory failure (shock), vascular blockage, glomerulonephritis, and obstruction to urine flow. Acute renal failure frequently arises as a complication of abdominal or vascular surgery. Of particular clinical importance are cases of acute renal failure associated with trauma, sepsis, postoperative complications, or medication, particularly antibiotics.
Post-operative complications such as infections, are overcome by the use of complex drugs such as antibiotics. Unfortunately, these same drugs can be toxic to the kidneys, particularly in elderly persons. Due to the increasing age of the hospital population and advances in complicated medical and surgical techniques, cases of acute renal failure are expected to increase in number and significance unless advances in treatment are made.
Treatment of acute renal failure typically involves dialysis, which removes the waste products and chemicals from the blood system. Despite some advances, the mortality rate associated with kidney disease still has not changed in many years. While dialysis provides a way to filter waste products and chemicals, the typical treatment regime poses a significant inconvenience to most patients. Usually treatment regimes involve lengthy time periods during which the patient is attached to the dialysis unit. The dialysis procedure is also repeated multiple times during a week. In many cases, the patient experiences side effects, such as muscle cramps and hypotension associated with the rapid change in the patient""s body fluid.
Kidney transplantation provides an alternative to dialysis. This involves replacing the patient""s kidney with a healthy kidney from a donor, if one becomes available. The implanted kidney then functions as the patient""s own kidney to filter blood and produce urine. Unfortunately, kidney rejection is a significant risk associated with transplantation, even with a good histocompatibility match. Immunosuppressive drugs such as cyclosporin and FK506 are usually given to the patient to prevent rejection. However, these immunosuppressive drugs have a narrow therapeutic window between adequate immunosuppression and toxicity. Prolonged immunosuppression can weaken immune systems, which can lead to a threat of infections developing. In some instances, even immunosuppression is not enough to prevent kidney rejection.
In an attempt to avoid the problems associated with dialysis and kidney transplantations, various methods have been reported in which the patients own kidney cells have been cultured in vitro. For example, U.S. Pat. No. 5,429,938 issued to Humes describes a method of reconstructing renal tubules using cultured kidney cells. The reconstructed renal tubules can be implanted into the patient.
Naughton et al. disclosed a three-dimensional tissue culture system in which stromal cells are laid over a polymer support system (see U.S. Pat. No. 5,863,531).
Vacanti et al. have disclosed methods for culturing cells in a three-dimensional matrix made of a biodegradable polymer. Organ cells are first cultured within the matrix and then implanted into the patient.
The above methods rely on shaping the support structure into the desired configuration of the organ. The correct three-dimensional configuration is essential for the reconstructed organ to function properly in vivo. Not only is the shape required to fit into the body cavity, but the shape also creates the necessary microenvironment for the cultured cells to grow and proliferate.
Therefore, a need exists for reconstructing artificial organs with the same three-dimensional infra-structure as the native organ. There is also a need to reconstruct an artificial organ for use as a permanent replacement of an organ.
The present invention provides compositions and methods for reconstructing artificial organs using a three-dimensional scaffold generated by decellularizing a natural biostructure. The three-dimensional scaffold is perfused with a population of cultured endothelial cells which attach to the three-dimensional scaffold and develop into an endothelial tissue layer. Continued growth and differentiation of the endothelial cells on the three-dimensional scaffold results in the formation of a primitive vascular system in the endothelial tissue layer. The primitive vascular system can then develop into a mature vascular system, and can also support the growth and development of additional cultured cell populations. The three-dimensional scaffold and the endothelial tissue layer with the primitive vascular system can be used to culture a variety of different cells and tissues in vitro and in vivo.
Accordingly, in one aspect, the invention features a method of reconstructing an artificial organ construct comprising:
perfusing a population of cultured endothelial cells into a three-dimensional scaffold formed by decellularizing a natural biostructure, such that endothelial cells attach to the three-dimensional scaffold;
culturing the endothelial cells in the three-dimensional scaffold until the endothelial cells produce an endothelial tissue layer comprising a primitive vascular system;
seeding at least one further second population of cultured cells into the three-dimensional scaffold such that the second cell population attaches to the endothelial tissue layer comprising the primitive vascular system and differentiates into a neomorphic organ structure.
During in vitro growth, the endothelial cells develop and produce an endothelial tissue layer comprising a primitive vascular system which envelopes the three-dimensional scaffold. The three-dimensional scaffold is composed of a biocompatible, non-degradable material. The endothelial tissue layer also provides a primitive vascular system that is capable of developing into a mature vascular system supports the growth and development of additional cultured cell populations. When grown in this three-dimensional scaffold, the proliferating cells mature and segregate properly to form tissues analogous to counterparts found in vivo.
The invention is based, in part, on the discovery that growth of endothelial cells in decellularized three-dimensional scaffolds will sustain active proliferation of additional cell populations. This may be due, in part, to the increased surface area of the natural biostructure-derived scaffold which permits in a prolonged period of active proliferation of endothelial cells. The prolonged proliferation enables the endothelial cells to develop to provide a primitive vascular system. The primitive vascular system subsequently provides support for the growth and development of additional cultured cell populations. In addition, the three-dimensionality of the decellularized biostructure allows for a spatial distribution which is the same as conditions in vivo, thus allowing for the formation of a microenvironment that is conducive for cellular maturation and migration. Optimal cell growth and development arises when the infrastructure of the microenvironment resembles the infra-structure of a natural organ. This provides the correct spacial distances that enable cell-cell interaction to occur. The growth of cells in the presence of this scaffold may be further enhanced by adding proteins, glycoproteins, glycosaminoglycans and a cellular matrix.
In one embodiment, the natural biostructure is an organ selected from the group consisting of heart, kidney, liver, pancreas, spleen, bladder, ureter and urethra. In another embodiment, the natural biostructure is a part of an organ selected from the group consisting of heart, kidney, liver, pancreas, spleen, bladder, ureter and urethra. In a preferred embodiment, the artificial organ construct is an artificial kidney construct. In another preferred embodiment, the three-dimensional scaffold is derived from a decellularized mammalian kidney. In another preferred embodiment, the endothelial cells are human endothelial cells. In another preferred embodiment, the second population comprises human kidney cells.
In another aspect, the invention features a method of treating a subject with an organ disorder comprising:
implanting a three-dimensional scaffold formed by decellularizing a natural biostructure perfused with a population of cultured endothelial cells, such that the endothelial cells attach to the three-dimensional scaffold to produce an endothelial tissue layer comprising a primitive vascular system, and at least one further second. population of cultured cells, such that the second cell population attaches to an endothelial tissue layer comprising a primitive vascular system and differentiates into a neomorphic organ structure; and
monitoring the subject for a modulation in the organ disorder.
In another aspect, the invention features an artificial organ construct comprising: a three-dimensional scaffold formed by decellularizing a natural biostructure, perfused with a population of cultured endothelial cells, such that the endothelial cells attach to the three-dimensional scaffold to produce an endothelial tissue layer comprising a primitive vascular system, and at least one further second population of cultured cells, such that the second cell population attaches to the an endothelial tissue layer comprising a primitive vascular system and differentiates into a neomorphic organ structure.
In another aspect, the invention features a method for reconstructing an artificial kidney construct comprising:
perfusing a population of cultured endothelial cells into a three-dimensional scaffold formed by decellularizing a mammalian kidney, such that endothelial cells attach to the three-dimensional scaffold;
culturing the endothelial cells in the three-dimensional scaffold until the endothelial cells produce an endothelial tissue layer comprising a primitive vascular system;
seeding a population of cultured kidney cells into the three-dimensional scaffold such that the kidney cell population attaches to the endothelial tissue layer comprising the primitive vascular system and differentiates into nephron structures.
In another aspect, the invention features a method of treating a subject with a kidney disorder comprising:
implanting a three-dimensional scaffold formed by decellularizing a mammalian kidney perfused with a population of cultured endothelial cells, such that the endothelial cells attach to the three-dimensional scaffold to produce an endothelial tissue layer comprising a primitive vascular system, and a population of cultured kidney cells, such that the kidney cell population attaches to the endothelial tissue layer comprising the primitive vascular system and differentiates into nephron structures; and monitoring the subject for a modulation in the kidney disorder.
In another aspect, the invention features an artificial kidney construct comprising:
a three-dimensional scaffold formed by decellularizing a mammalian kidney perfused with a population of cultured endothelial cells, such that the endothelial cells attach to the three-dimensional scaffold to produce an endothelial tissue layer comprising a primitive vascular system, and a population of cultured kidney cells, such that the kidney cell population attaches endothelial tissue layer comprising the primitive vascular system and differentiates into nephron structures.
In another aspect, the invention features a method for screening a compound that modulates kidney cells comprising:
providing an artificial kidney construct with a three-dimensional scaffold formed by decellularizing a mammalian kidney, perfused with a population of cultured endothelial cells, such that the endothelial cells attach to the three-dimensional scaffold to produce an endothelial tissue layer comprising primitive vascular system, and a population of cultured kidney cells, such that the kidney cell population attaches to the endothelial tissue layer comprising the primitive vascular system and differentiates into nephron structures;
contacting the artificial kidney construct with a library of test compounds;
selecting from the library of test compounds a compound of interest that modulates kidney cells.
In one embodiment, the modulator is cytotoxic to the kidney cells. In another embodiment, the modulator is therapeutic to the kidney cells. In one embodiment, the compound is a chemical agent, or a pharmaceutical agent.
In another aspect, the invention features a method for processing an aqueous solution comprising:
providing an artificial kidney construct having a three-dimensional scaffold formed by decellularizing a mammalian kidney perfused with a population of cultured endothelial cells, such that the endothelial cells attach to the three-dimensional kidney scaffold to produce an endothelial tissue layer comprising a primitive vascular system, and a population of cultured kidney cells, such that the kidney cell population attaches to endothelial tissue layer comprising the primitive vascular system and differentiates into nephron structures;
delivering the aqueous solution to the luminal side of the artificial kidney construct;
collecting a processed aqueous solution from the abluminal side of the artificial kidney construct.
In one embodiment, the aqueous solution is unfiltered blood and the processed aqueous solution is filtered blood.